Activity of ceftolozane/tazobactam and imipenem/relebactam against Gram-negative clinical isolates collected in Mexico—SMART 2017–2021

Abstract Objectives To investigate the activities of ceftolozane/tazobactam and imipenem/relebactam against Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa isolated from hospitalized patients in Mexico in 2017–2021. Methods MICs were determined by CLSI broth microdilution and interpreted using CLSI M100 breakpoints. β-Lactamase genes were identified in ceftolozane/tazobactam-, imipenem/relebactam-, and/or imipenem-non-susceptible isolates. Results Ceftolozane/tazobactam and imipenem/relebactam inhibited 89% and 99% of E. coli isolates (n = 2337), and 87% and 94% of K. pneumoniae isolates (n = 1127). Sixty-four percent of E. coli and 47% of K. pneumoniae had an ESBL non-carbapenem-resistant Enterobacterales (ESBL non-CRE) phenotype. Eighty-six percent and 91% of ESBL non-CRE E. coli and K. pneumoniae were ceftolozane/tazobactam susceptible, and 99.9% and 99.8% were imipenem/relebactam susceptible. Ceftolozane/tazobactam was the most active agent studied against P. aeruginosa (n = 1068; 83% susceptible), 9–28 percentage points higher than carbapenems and comparator β-lactams excluding imipenem/relebactam (78% susceptible). Ceftolozane/tazobactam remained active against 35%–58%, and imipenem/relebactam against 32%–42%, of P. aeruginosa in meropenem-, piperacillin/tazobactam-, and cefepime-non-susceptible subsets. The majority of isolates of ceftolozane/tazobactam-non-susceptible E. coli carried an ESBL, whereas among ceftolozane/tazobactam-non-susceptible K. pneumoniae and P. aeruginosa, the majority carried carbapenemases. The most prevalent carbapenemase observed among E. coli (estimated at 0.7% of all isolates), K. pneumoniae (4.8%) and P. aeruginosa (10.0%) was an MBL. Almost all imipenem/relebactam-non-susceptible E. coli and K. pneumoniae carried MBL or OXA-48-like carbapenemases, whereas among imipenem/relebactam-non-susceptible P. aeruginosa, 56% carried MBL or GES carbapenemases. Conclusions Ceftolozane/tazobactam and imipenem/relebactam may provide treatment options for patients infected with β-lactam-non-susceptible Gram-negative bacilli, excluding isolates carrying an MBL- or OXA-48-like carbapenemase.


Introduction
ESBL-producing and carbapenem-resistant Enterobacterales (CRE), and carbapenem-resistant Pseudomonas aeruginosa are frequently MDR and leave clinicians with few or no safe and effective treatment choices.[3] Ceftolozane/tazobactam, an antipseudomonal cephalosporin combined with a longstanding β-lactamase inhibitor, is a newer antimicrobial agent approved in Mexico for the treatment of complicated urinary tract and intra-abdominal infections, and hospital-acquired and ventilator-associated bacterial pneumonia (HAP/VAP). 4Imipenem/relebactam is a combination of imipenem/ cilastatin (carbapenem/renal dehydropeptidase inhibitor) with relebactam, a non-β-lactam diazabicyclooctane inhibitor of class A and C β-lactamases, 5,6 that is approved by the US FDA for the treatment of complicated urinary tract and intra-abdominal infections, and HAP/VAP, 7 and by the EMA for the treatment of infections due to aerobic Gram-negative organisms in adults with limited treatment options; for the treatment of HAP, including VAP, in adults; and for the treatment of bacteraemia that occurs in association with, or is suspected to be associated with, HAP or VAP, in adults. 8Imipenem/ relebactam is not approved currently for clinical use in Mexico.
Published surveillance data for ceftolozane/tazobactam and imipenem/relebactam tested against clinical isolates of Enterobacterales and P. aeruginosa from Mexico are very limited. 9,10Therefore, in the current study, we evaluated the activity of ceftolozane/tazobactam, imipenem/relebactam, and nine comparator agents against common Gram-negative pathogens (Escherichia coli, Klebsiella pneumoniae, P. aeruginosa) collected in Mexico by the SMART (Study for Monitoring Antimicrobial Resistance Trends) global surveillance programme and also identified β-lactamase resistance mechanisms in phenotypically resistant isolate subsets.

Bacterial isolates
From 2017 to 2021, eight clinical laboratory sites in Mexico participated in the SMART global surveillance programme; six of the eight sites participated in all five years, one additional site participated in 2019, and one additional site participated in 2021.Sites were each asked to collect consecutive, clinically significant isolates of aerobic or facultatively anaerobic Gram-negative bacilli from intra-abdominal infection (IAI; 75 isolates in 2017 and 50 isolates/year in 2018-2021), lower respiratory tract infection (LRTI; 100 isolates/year), urinary tract infection (UTI; 75 isolates in 2017 and 50 isolates/year in 2018-2021), and bloodstream infection (BSI; 50 isolates/year in 2018-2021 only) samples.Isolates were restricted to one isolate per patient per Gram-negative species per year.Organismspecific quotas are not used in the collection of isolates by the SMART global surveillance programme.All isolates were shipped to IHMA (Schaumburg, IL, USA) where organism identity was confirmed using MALDI-TOF MS (Bruker Daltonics, Billerica, MA, USA), and antimicrobial susceptibility and molecular testing was performed.

Antimicrobial susceptibility testing
MICs were determined by the CLSI reference broth microdilution method 11 on custom-made dehydrated broth microdilution panels manufactured by TREK Diagnostic Systems (Thermo Fisher Scientific, Oakwood Village, OH, USA) in 2017 and frozen broth microdilution panels prepared at IHMA in 2018-2021.MICs were interpreted using 2023 CLSI M100 breakpoints. 12An ESBL non-CRE phenotype was defined for E. coli and K. pneumoniae as an isolate testing as non-susceptible to ceftriaxone (MIC ≥2 mg/L) and susceptible to ertapenem (MIC ≤0.5 mg/L).For E. coli and K. pneumoniae, the EUCAST breakpoint for amikacin 13 was used to interpret MIC data for that agent because the concentration range tested did not extend low enough to include the revised 2023 CLSI susceptible breakpoint for that agent. 12

Screening for β-lactamase genes
5][16] All isolates from 2017 to 2019 that met the screening criteria were tested.Isolates characterized in 2020 and 2021 were a random sample of those that met the screening criteria.In total, 274 of 287 (95.5%)Enterobacterales isolates that qualified for molecular characterization in 2020 and 2021 were characterized, as were 185 of 238 (77.7%) qualified P. aeruginosa isolates.The percentage of characterized, qualifying isolates collected in 2020 and 2021 was factored into the estimation of carbapenemase rates.

Results
A total of 4880 isolates of Enterobacterales and 1068 isolates of P. aeruginosa were collected by the eight laboratories from 2017 to 2021 (Table S1, available as Supplementary data at JAC-AMR Online).E. coli (n = 2337) and K. pneumoniae (n = 1127) were the two most common species of Gram-negative bacilli isolated and accounted for 71% of the 4880 isolates of Enterobacterales.Together, E. coli, K. pneumoniae and P. aeruginosa (the third most frequently isolated species of Gram-negative bacilli) accounted for 67% of all Gram-negative bacilli collected by the SMART global surveillance programme in Mexico from 2017 to 2021.Among E. coli, K. pneumoniae and P. aeruginosa isolates, 13.5%, 37.3% and 51.6%, respectively, were collected from patients with LRTI; 35.5%, 26.0% and 17.7% from patients with UTI; 33.5%, 16.8% and 17.6% from patients with IAI; and 17.5%, 19.9% and 12.7% from patients with BSI (Table S1).An infection source was not identified for 0.1% of E. coli, 0.1% of K. pneumoniae and 0.4% of P. aeruginosa isolates.
Ceftolozane/tazobactam and imipenem/relebactam were active against 89% and 99% of E. coli isolates, respectively, and against 87% and 94% of K. pneumoniae isolates (Table 1).Imipenem, meropenem and ertapenem percent susceptible values ranged from 97% to 99% for E. coli and from 91% to 93% for K. pneumoniae.The addition of relebactam to imipenem had only a minor impact on percent susceptible values for both E. coli (0.5%) and K. pneumoniae (1.4%).
Among E. coli and K. pneumoniae, 64% and 47% of isolates, respectively, had an ESBL non-CRE phenotype resulting in overall percent susceptible values of <50% for cefepime, ceftazidime and ceftriaxone.Only 61% of all K. pneumoniae isolates, 51% of all E. coli isolates, 34% of ESBL non-CRE K. pneumoniae isolates, and 27% of ESBL non-CRE E. coli isolates had a cefepimesusceptible or cefepime susceptible-dose dependent phenotype.Percent susceptible values for ESBL non-CRE phenotype E. coli and K. pneumoniae were 86% and 91% for ceftolozane/tazobactam and 99.9% and 99.8% for imipenem/relebactam.Only 68% of ESBL non-CRE phenotype E. coli and 48% of ESBL non-CRE phenotype K. pneumoniae were piperacillin/tazobactam-susceptible.Thirty percent or less of ESBL non-CRE phenotype E. coli and K. pneumoniae were levofloxacin-susceptible.As expected, the addition of relebactam to imipenem had only a minor impact on the percent susceptible value (0.4% or less) for ESBL non-CRE phenotype isolates of E. coli and K. pneumoniae as susceptibilities to imipenem alone were ≥99.4%.Percent susceptible values for ceftolozane/tazobactam ranged from 85% (LRTI) to 90% (BSI, IAI and UTI) among E. coli specimen sources and from 83% (BSI) to 91% (IAI) among K. pneumoniae specimen sources (Table S2).Percent susceptible values for imipenem/relebactam had narrower ranges than ceftolozane/tazobactam, from 98% (LRTI) to >99% (IAI and UTI) for E. coli specimen sources, and from 92% (BSI) to 95% (IAI) for K. pneumoniae specimen sources.Amikacin percent susceptible values for ESBL non-CRE phenotype Karlowsky et al.E. coli and K. pneumoniae were ∼10% lower than for imipenem/ relebactam, imipenem, meropenem and ertapenem.

Discussion
Published studies describing in vitro surveillance data for ceftolozane/tazobactam tested against clinical isolates of Enterobacterales or P. aeruginosa from Mexico or countries in Latin America are very limited. 10The current study expands upon our previous publication describing the in vitro data for imipenem/relebactam tested against clinical isolates of Enterobacterales and P. aeruginosa from Latin American We observed that ceftolozane/tazobactam inhibited 89% of E. coli and 87% of K. pneumoniae isolates (Table 1).An ESBL phenotype was frequently seen among both E. coli (64%) and K. pneumoniae (47%) in Mexico resulting in overall percent susceptible values of <50% for cefepime, ceftazidime and ceftriaxone.8][19] Ceftolozane/tazobactam was considerably more active than piperacillin/tazobactam (by 15 to 23 percentage points) against both E. coli (74% piperacillin/ tazobactam-susceptible) and K. pneumoniae (64%) (Table 1).Ceftolozane/tazobactam percent susceptible values were also >18% higher for ESBL non-CRE phenotype E. coli (86% susceptible versus 68%) and K. pneumoniae (91% versus 48%) than values for piperacillin/tazobactam.A smaller study of 69 E. coli and 15 K. pneumoniae isolates collected in Mexico in 2016-2017 and tested against ceftolozane/tazobactam reported similar results to ours for all isolates tested and for isolates with an ESBL non-CRE phenotype. 10Cumulatively, these data argue strongly against the empirical use of piperacillin/tazobactam, cefepime, ceftazidime and ceftriaxone to treat a serious infection known or suspected to be caused by E. coli, K. pneumoniae or other less common Enterobacterales pathogens in Mexico without review of an isolate-specific antibiogram.
Seventy-seven percent (187/243) of ceftolozane/tazobactamnon-susceptible E. coli were shown to carry an ESBL as their only β-lactamase, 10% (24/243) carried an MBL or an OXA-48-like carbapenemase, 9% (23/243) carried AmpC (22 were CMY-type and one was MIR-type) with or without an ESBL, and 4% (9/243) of isolates did not have an acquired β-lactamase gene identified (Table 3).In contrast, the majority of ceftolozane/tazobactamnon-susceptible K. pneumoniae isolates (57%, 82/144) carried a carbapenemase gene (mostly an MBL or OXA-48-like carbapenemase).The 5%-6% difference observed between E. coli and K. pneumoniae in the percentages of isolates susceptible to imipenem/relebactam and the carbapenems (Table 1) is explained by a similar difference in the percentage of E. coli and K. pneumoniae isolates that carried an MBL or OXA-48-like carbapenemase gene (Table 2).Relebactam is inactive against Ambler class B (MBLs) and class D carbapenemases (e.g.OXA-48-like) and would not be expected to augment the activity of imipenem against isolates carrying these carbapenemases. 6eftolozane/tazobactam was the most active agent studied against P. aeruginosa, inhibiting 83% of isolates (Table 1).An earlier, smaller study of 127 P. aeruginosa isolates collected in Mexico in 2016-2017 reported a 19% lower percent susceptible value (64%) than our study; however, the isolates in the former study were also 17% to 22% less susceptible to meropenem (45%), piperacillin/tazobactam (50%) and ceftazidime (50%) than the isolates we tested and details of isolate collection and inclusion were not provided. 10In the current study, imipenem/relebactam (78% susceptible) was 5% less active than ceftolozane/tazobactam.However, a previous study reported that imipenem/relebactam remained active against a subset of ceftolozane/tazobactamnon-susceptible isolates, depending upon the β-lactamases and other resistance mechanisms present in the isolates, suggesting that both agents should be tested against clinical isolates, if possible. 20Specifically, the earlier study observed that in isolates of P. aeruginosa without a detected non-intrinsic β-lactamase gene, the presence of both a PDC (Pseudomonas-derived cephalosporinase) mutation and an indicator of PDC up-regulation frequently resulted in an imipenem/relebactam-susceptible/ceftolozane/ tazobactam-non-susceptible phenotype. 20A second study confirmed the finding that ceftolozane/tazobactam-non-susceptible P. aeruginosa isolates without detected non-intrinsic β-lactamase genes were associated with mutations in PDC in combination with PDC up-regulation. 21Percent susceptible values for carbapenems, third-and fourth-generation cephems, piperacillin/tazobactam, and levofloxacin against P. aeruginosa were all compromised in the current study, ranging from 55% to 74% susceptible.
In the current study, imipenem, meropenem and ertapenem percent susceptible values ranged from 97% to 99% for E. coli (>99% for ESBL non-CRE isolates) and from 91% to 93% for K. pneumoniae (>99% for ESBL non-CRE isolates) (Table 1).Earlier data for Enterobacterales isolates collected in Mexico from 2005 to 2015 showed comparable meropenem and imipenem percent susceptible values for E. coli (95% to >99%) but higher percent susceptible values for K. pneumoniae (95%-99%). 19,22,23In one report, 33% of Enterobacterales isolates from Mexico in 2012-2015 (n = 1862) were ESBL-positive and only 0.8% of isolates carried a carbapenemase. 22We estimated that 1.3% of E. coli, 7.5% of K. pneumoniae and 13.0% of P. aeruginosa isolates infecting hospitalized patients in Mexico in 2017-2021 carried a carbapenemase (Table 2), suggesting an increase in carbapenemase carriage among Enterobacterales over time.MBLs were the most prevalent carbapenemase type observed among E. coli (0.7% of all E. coli isolates), K. pneumoniae (4.8%) and P. aeruginosa (10.0%).In P. aeruginosa, the rate in the current study (10.0%) is double that reported in an earlier study from 2015 to 2017 (4.8% MBL among 524 isolates) from Mexico. 24Imipenem/ relebactam percent susceptible values for E. coli and K. pneumoniae correlated closely with the presence of MBLs and OXA-48-like carbapenemases in the current study.
Seventy-eight percent of P. aeruginosa isolates in the current study were imipenem/relebactam-susceptible compared with percent susceptible values of 55% for imipenem and 62% for meropenem (Table 1).A previous report of 1794 P. aeruginosa isolates collected in hospitals in Mexico in 2012-2015 similarly reported that 65% of isolates were meropenem-susceptible. 22ifty-six percent of characterized imipenem/relebactam-nonsusceptible P. aeruginosa in the current study carried an MBL or GES carbapenemase; ESBLs were only identified in 10% of isolates and no acquired β-lactamase was detected in 33% of isolates (Table 3).In the isolates with ESBLs only or where no acquired β-lactamase gene was detected, OprD loss/mutation in combination with PDC derepression likely contributed to the imipenem/ relebactam-non-susceptible phenotypes. 25Imipenem is known to be a strong inducer of PDC, and PDC expression levels have been correlated to imipenem MICs in P. aeruginosa with defects in OprD. 26mipenem/relebactam activity against P. aeruginosa is unaffected by efflux-based resistance and is less affected by OprD loss than imipenem alone. 6Imipenem/relebactam also retains in vitro activity against isolates with KPC or PDC mutations that result in resistance to ceftazidime/avibactam or ceftolozane/tazobactam. 27 Amikacin inhibited 91% of E. coli, 90% of K. pneumoniae, and 80% of P. aeruginosa using EUCAST breakpoints (Table 1).Even though it appears highly active in vitro, use of amikacin and other aminoglycosides is widely discouraged in clinical guidelines and by CLSI and EUCAST laboratory in vitro testing standards as all are associated with major toxicity and therapeutic limitations. 12,13,28he strengths of the current study are that it collected isolates from the same six sites in Mexico according to a consistent protocol for 5 years and used reference broth microdilution antimicrobial susceptibility testing and molecular testing performed in a central laboratory.Study limitations include that the number of medical centres participating was limited; sample quotas were used to collect isolates from different infection types that may affect the overall estimates of resistance and β-lactamase prevalence; isolates susceptible to ceftolozane/tazobactam and imipenem/ relebactam were not molecularly characterized; and an ESBL non-CRE phenotype was defined as an isolate of E. coli and K. pneumoniae testing as non-susceptible to ceftriaxone (MIC ≥2 mg/L) and susceptible to ertapenem (MIC ≤0.5 mg/L) and did not include clavulanic acid-based phenotypic confirmatory testing. 12ased on our in vitro data, we conclude that ceftolozane/ tazobactam and imipenem/relebactam may provide important treatment options for many patients infected with β-lactamnon-susceptible Gram-negative bacilli, including ESBL non-CRE E. coli and K. pneumoniae and β-lactam-non-susceptible P. aeruginosa.Increases in the prevalence of MBL-positive isolates of Gram-negative bacilli have been documented in Mexico and in Latin America and will erode the activities of all newer β-lactam/ β-lactamase inhibitor combinations, including ceftolozane/tazobactam and imipenem/relebactam.Ongoing surveillance of the in vitro activities of established and newer antimicrobial agents against Gram-negative pathogens and monitoring for the spread of β-lactamase genes, particularly for MBLs, is critical.

Table 1 .
Antimicrobial susceptibility of all clinical isolates of E. coli, K. pneumoniae and P. aeruginosa and resistant phenotype subsets collected by the SMART global surveillance programme in Mexico from 2017 to 2021 For E. coli and K. pneumoniae, the EUCAST breakpoint for amikacin was used because the concentration range tested did not extend low enough to include the revised 2023 CLSI susceptible breakpoint for that agent.CRE phenotype was defined by an isolate testing non-susceptible to ceftriaxone (MIC ≥2 mg/L) and susceptible to ertapenem (MIC ≤0.5 mg/L).

Table 2 .
Estimated carbapenemase rates among E. coli, K. pneumoniae and P. aeruginosa isolates collected by the SMART global surveillance programme in Mexico from 2017 to 2021 countries, including Mexico.The current study tests and describes a more current (2017-2021), larger isolate dataset from hospitalized patients in Mexico, includes testing of ceftolozane/tazobactam, and offers more in-depth analyses than our earlier publication.

Table 3 .
Acquired β-lactamases detected in ceftolozane/tazobactam-non-susceptible and imipenem/relebactam-non-susceptible E. coli, K. pneumoniae and P. aeruginosa a collected in Mexico by SMART from 2017 to 2021 Pseudomonas-derived cephalosporinase) is not shown in this analysis.bNoacquired β-lactamases included in the screening algorithm were detected.
a Intrinsic AmpC found in P. aeruginosa (PDC,